Autoflex 2 smartbeam

The sample (1 μL, approx. 1 mg/mL), and the matrix alpha-cyano-4-hydroxycinnamic acid (1 μL, 10 mg/mL in a 1:1 mixture of water and acetonitrile, with 1% trifluoroacetic acid) were spotted onto a 384 spot ground steel target, mixed and left to dry. A Bruker autoflex II smartbeam (Bruker Daltonics GmbH, Leipzig, Germany) with MALDI laser source (355 nm, 200 Hz) was used for all measurements. Measurements were performed in linear, positive mode, up to 1000 scans were accumulated and a mass range up to m/z 200000 was observed. The manufacturer’s software suite (flexControl and flexAnalysis), as well as Origin (OriginLab Corp., MA, USA ) were used for data acquisition and evaluation. The MALDI-TOF MS spectra of the six tested proteins are shown in Figure S7 (see Electronic Supplementary Material).

All measurements were performed using a Bruker Autoflex II Smartbeam™ in linear mode. A mixture of two PMMA polymers with different peak molecular weights (Mp = 1960 and Mp = 5050) was used as a calibrant. The compounds were dissolved in tetrahydrofuran (THF) to form a 3 mg/mL solution. A total of 20 µL of this solution was mixed with 50 µL of a 20 mg/mL trans-2-(3-(4-tert-butylphenyl)-2-methyl-2-propenylidene)malononitrile (DCTB) solution in THF. A total of 0.5 µL of the resulting mixture was deposited next to each sample spot. A calibration file (see Table S1, Supplementary Materials) with reference masses for the n-mers of PMMA was then used to (re)calibrate the instrument before measuring each sample.

All four scans were merged in the ImageJ software (1.51, National Institute of Health, USA) and analyzed. The fluorescence intensity was measured for 9 pixels (225 µm²) as the region of interest (ROI) and the mean value was used. The chip coordinates of the hits were used for the spatial resolution in the MALDI-TOF-MS instrument (Bruker autoflex II smartbeam™). The peptides were cleaved from the resin in an ammonia gas atmosphere overnight. Afterward, the 1.5-DHAB matrix (6 ml of 4:1 EtOH: ultrapure water, 30 mg 1.5-DHAB, 10.5 mg ammonium citrate dibasic) was applied to the chip via an airbrush system. Finally, the chip was measured with a MALDI-TOF-MS.